In a process referred to as quorum sensing, bacteria communicate using chemical signal molecules called autoinducers. By monitoring increases and decreases in autoinducer concentration, quorum-sensing bacteria track changes in cell-population density and synchronously switch into and out of group behaviors. Quorum sensing allows bacteria to collectively carry out tasks that would be unsuccessful if carried out by an individual bacterium acting alone.
Gram-negative quorum-sensing bacteria typically employ acyl homoserine lactones (AHLs) as autoinducers and each bacterial species detects the cell-density-dependent accumulation of one or more particular AHL molecule(s). AHL detection occurs by one of two distinct mechanisms.
In the first mechanism, cytoplasmic LuxR-type proteins detect AHLs that diffuse into the cell (FIG. 1A) (Fuqua et al., 1994). In these cases, the unliganded LuxR-type proteins are insoluble and degraded at low cell density (LCD) when AHL concentrations are low (Zhu and Winans, 1999, 2001). At high cell density (HCD), when AHLs are present, AHL binding to the cognate LuxR-type proteins promotes folding of the LuxR-type protein-AHL complexes. The complexes bind DNA and activate quorum-sensing target genes (Zhang et al, 2002). However, there are a few cases in which the LuxR-type proteins fold, bind DNA, and repress target gene transcription in the absence of AHL (Minogue et al., 2002, 2005; Sjoblom et al., 2006). In these cases, accumulation and binding of AHL relieves repression and this depends on the location of the DNA binding site, rather than on some unique structural feature of the receptor. Chromobacterium violaceum provides an example of this first mechanism, with the AHL being C6-homoserine lactone (HSL), the LuxR-type protein being CviR, and the target genes being vio genes, which are involved in production of violacein protein.
In the second mechanism, AHLs are detected by membrane-bound two component histidine kinase-type proteins of the LuxN family (FIG. 1B) (Freeman et al., 2000; Jung et al., 2007; Swem et al., 2008; Timmen et al., 2006). In these cases, accumulated AHLs are detected outside the cell (e.g. in the periplasm) and AHL binding by the cognate LuxN receptor elicits a change in its auto-phosphorylation and phospho-transfer activities. These complexes relay information internally by phosphorylation cascades that impinge on downstream DNA binding proteins that are responsible for directing gene expression changes. This causes a change in the phosphorylation state of a downstream DNA binding transcription factor, which alters its activity and promotes the quorum-sensing gene expression response.
Infectious bacteria, which include human, animal, plant, and marine pathogens, use AHL quorum sensing strategies to control virulence. Typically, bacterial infections are treated with bactericidal or bacteriostatic molecules that impede four major processes: DNA replication, transcription, translation or tetrahydrofolic acid synthesis. Existing methods for treating bacterial infection unfortunately exacerbate the growing antibiotic resistance problem because they inherently select for growth of bacteria that in turn can resist the drug. What is needed are new treatments that avoid selecting for drug resistant bacteria.
It is well established that quorum sensing plays a fundamental role in bacterial pathogenicity in both Gram-positive and Gram-negative bacteria. However, previous attempts to inhibit AHL-mediated quorum sensing in Gram-negative bacteria have not provided promising results in vivo.
The bacterium Pseudomonas aeruginosa is the major pathogen associated with cystic fibrosis lung infection, keratitis eye infection, and third-degree burn-associated skin infections. There are P. aeruginosa mutant strains that lack the AHL synthase and thus do not produce endogenous autoinducer. Molecules have been studied in vitro that inhibit LasR, a receptor for the AHL. However, those studies on P. aeruginosa have not included in vivo testing on wild type bacteria.
Quorum sensing also controls biofilm formation. Biofilms are communities of bacterial cells adhered to surfaces and are highly problematic, for example in industrial processes (e.g., clogging of cooling towers in manufacturing plants) and in hospital or other clinical settings (e.g., catheter and implant infections). Initial studies with Staphylococcus aureus and Staphylococcus epidermidis indicated that manipulation of a form of quorum sensing that is peptide-mediated would not have successful results. Most notably, disruption of the peptide quorum-sensing circuit in S. epidermidis by deleting necessary quorum sensing genes led unexpectedly to increased biofilm formation on implanted medical devices. Therefore what is needed are new treatments for bacterial infection that can more subtly manipulate bacterial behaviors that promote health problems.